In-vitro Activity of 10 Antifungal Agents against 320 Dermatophyte Strains Using Microdilution Method in Tehran.

Dermatophyte fungi are the etiologic agents of skin infections commonly referred to as ringworm. These infections are not dangerous but as a chronic cutaneous infections they may be difficult to treat and can also cause physical discomfort for patients. They are considered important as a public health problem as well. No information is available regarding the efficacy of antifungal agents against dermatophytes in Tehran. Therefore, in this study we evaluated the efficacy of 10 systemic and topical antifungal medications using CLSI broth microdilution method (M38-A). The antifungal agents used included griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole, voriconazole, clotrimazole, ciclopirox olamine, amorolfine and naftifine.Fifteen different species of dermatophytes which were mostly clinical isolates were used as follows; T. mentagrophytes, T. rubrum, E. floccosum, M. canis, T. verrucosum, T. tonsurans, M. gypseum, T. violaceum, M. ferruginum, M. fulvum, T. schoenleinii, M. racemosum, T. erinacei, T. eriotrephon and Arthroderma benhamiae. The mean number of fungi particles (conidia) inoculated was 1.25 ×10⁴ CFU/mL. Results were read after 7 days of incubation at 28 °C. According to the obtained results,itraconazole and terbinafine showed the lowest and fluconazole had the greatest MIC values for the most fungi tested. Based on the results, it is necessary to do more research and design a reliable standard method for determination of antifungal susceptibility to choose proper antibiotics with fewer side effects and decrease antifungal resistance and risk of treatment failure.


Introduction
Cutaneous mycoses are divided into two groups based on their etiologic agent, including dermatomycosis and dermatophytosis. Dermatomycosis are cutaneous mycoses caused by any fungi other than dermatophytes. Dermatophytes are a closely related group of fungi in 3 anamorphic genera of Trichophyton, Microsporum and Epidermophyton that originate from humans, different animals or soil. Except for a few systemic diseases and immunosuppressed patients (1-7), they usually invade only keratinized tissue, such as skin epidermis, nail plate and hair. Although there are approximately more than 40 different species in these three genera, most infections are due to Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum canis. A single species may be seen in several disease types.
Griseofulvin was the only approved systemic antidermatophytic agent, however,at present new antifungal drugs with fewer side effects is available. Terbinafine (allylamines), ketoconazole (imidazole group), itraconazole, and fluconazole (triazoles group) are mostly used orally, and clotrimazole (imidazole), ciclopir ox olamine (substituted pyridine), amorolfine (morpholin) and naftifine (allylamines) are usually applied topically for the treatment of these infections. Treatment failure occurs in 25-40% of patients with onychomycosis and 6.5% of those with tineacorporis and tinea cruris (8). Although, role of drug resistance in treatment failure is not clearly known, in-vitro susceptibility testing could help the clinicians to select the proper antifungal agent.There is no standard method available for susceptibility testing of dermatophytes. Determination of MIC (Minimal Inhibitory Concentration) by CLSI dilution method (M38-A) has been suggested in several studies (3,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). There are more than 33 articles published on epidemiology, clinical aspects and etiology of dermatophytosis from Iran. However, only few articles studing antifungal susceptibility with the different method for these fungi (19)(20)(21)(22)(23)(24). According to CLSI protocol M38-A, completely synthetic medium RPMI1640 (without sodium bicarbonate and L-glutamine at pH 7.0) supplemented with 0.165M morpholinepropanesuphonic acid (MOPS) is suitable for broth dilution based antifungal susceptibility determination for filamentous fungi (15). However, there are differences in time and incubation periods, form and numbers of inoculated fungi and MIC breakpoint determination and culture media used for different experiments.
Correlations of MICs with clinical outcomes have not been determined (10). It seems that comparing relying on the crude results is difficult, thus biostatic methods offered calculation of some parameters, like geometric mean (GM). Definitely, the geometric mean is the nth root of products of the value in a series of n-values. The geometric mean is more useful and representative than the arithmetic mean when describing a series of reciprocal or fractional values. The geometric mean can be used only for positive values (25).
The purpose of this study was to determine the susceptibility of 10 oral and topical antifungal agents including fluconazole, itraconazole, ketoconazole, voriconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine and griseofulvin against clinical dermatophytes isolated from patients in Tehran, Iran.

Experimental
Dermatophyte strain A total of 320 dermatophyte strains belonging to 15 species were recognized using conventional methods such as slide culture, growth in CMA with 1% glucose, sabouraud dextrose agar with 3% NaCl, hair perforation, growth at 37°C, urease activity, and Trichophyton agar. T. eriotrephon, and Arthroderma benhamiae were diagnosed based on sequencing.

Inoculum preparation
Stock inoculum suspensions of the fungi were prepared from 7 to 10 day old cultures grown on PDA at 28°C. Mature colonies were covered with approximately 5 mL of sterile saline (0.85%) by scraping the surface with the tip of a sterile swab. The resulting mixture of conidia and hyphal fragments was withdrawn and transferred to sterile tubes. Heavy particles were allowed to sediment for 15 to 20 min at room temperature. The upper suspension was transferred to another sterile tube and mixed with a vortex mixer for 15 sec. Before the turbidity of the supernatants was measured spectrophotometrically (Jenway Model 6305) at a wavelength of 530 nm, transmission was adjusted to 65 by ETO sterilized disposable cuvette. Each suspension was diluted 1:50 to 1: 100 in RPMI 1640 to obtain twice the final test inoculum concentration. The inocula corresponding to the strains were quantified by plating 5 µL of a 1:100 dilution of the adjusted inoculum on PDA plates and spread in 4 directions by a sterile loop. The plate contents were incubated at 30°C and observed daily for the presence of fungal colonies. Colonies were counted, when the growth became visible, as number of CFU per milliliter.
Test procedure All the tests were performed in sterile, flat-bottomed, 96-well micro plates (Orange Scientific, E.U). Aliquots of 100 µL of the 2× drug dilutions were inoculated into the wells with a multichannel pipette. The micro plates were put in storage at -22°C until used.
For performing the susceptibility testing, 100 µL of the diluted inoculum suspensions was added to each well to bring the drug dilutions to the final test concentrations. Growth and sterility control wells were also prepared for each isolate tested. The micro plate contents were incubated at 28 °C, avoiding desiccation of the wells, and were read visually with the aid of an inverted reading mirror after 7 days of incubation. Every test was repeated at least twice.

Reading results
For fluconazole and griseofulvin, the MIC was defined as approximately 50% growth inhibition compared to the growth control well. For other antifungal agents, the MIC was defined as the lowest concentration showing 100% growth inhibition.

Data analysis
Geometric mean, MIC range, MIC50, MIC80 and MIC90 were obtained for all the isolates tested. MIC value of antifungal drugs for different species were compared by repeated measure and one-way ANOVA and post-hoc Scheffe tests using SPSS version 16 software. P-values < 0.05 were considered statistically significant.

Results and Discussion
A total of 320 dermatophytes, mostly clinically isolated from patients, were tested using modified M38 -A CLSI micro dilution method. T. mentagrophytes, followed by T. rubrum and E. floccosum, were the most common dermatophytes isolated from patients in Tehran.
In-vitro activities of 10 antifungal agents that were obtained for all the isolates tested. Geometric mean (GM) of itraconazole and terbinafine was lowest for T. rubrum, T. mentagrophytes and E. floccosum indicating that these drugs are most potent in in-vitro studies. Mean MICs of antifungal drugs did not show statistically significant differences between different species (p > 0.05).Voriconazole was more effective than ketoconazole, however fluconazole and griseofulvin had the lowest MIC against these 3 dermatophytes. Amongst topical antifungal agent, clotrimazole showed lowest MIC value followed by amorolfine, naftifine and ciclopirox olamine.
Itraconazole and terbinafine showed the lowest GM MIC for M. canis.Griseofulvin had GM MIC lower than voriconazole, followed by ketoconazole and fluconazole against this microorganism. After fluconazole, griseofulvin had the highest GM MIC for mostother dermatophytes.Between 4 topicalantifugal agents, clotrimazole had the lowest MIC and GM MIC. Amorolfinewas the second antifungal agent against M. canis, and naftifine and ciclopirox olamine were rated third and fourth respectively.
Although terbinafine had the greatest in-vitro activity and lowest GM MIC against T. tonsurans, M. gypseum, Arthroderma benhamiae, voriconazole had the lowest MIC against T. violaceum. A surprising result was that itraconazole was the most potent antifungal agent against most of the microorganisms tested in this study. These tests were performed under variable conditions in terms of type of culture media for sporulation, incubation temperature, number and type of inoculated fungi and method, reading and interpreting the result by different researchers.
Thus it would be difficult to compare the results.
The culture media used for sporulation in this study was the PDA media which was in according with many other studies like Li et al., Santos et al.,Sarifakioglu et al.,Galuppi et al.,Ghannoum et al,Carrillo et al,Fernandez Torres et al,and Esteban et al. studies (8,10,12,14,16,[26][27][28][29]. A 7-10-day incubation time was allocated for sporulation of the majority of dermatophytes, like T. mentagrophytes and T. rubrum. Longer periods up to 6 weeks were required for other fungi, especially T. schoenleinii and T. violaceum until the colony morphology changed to a white fluffy appearance. The incubation time used in this study was similar to the one used by Barro set et al. (30), Santos et al. (27), and Fernandez Torrez et al. (12,14).
For inoculation of fungal suspension into the 96 well microplates containing antibiotics, the suspension was diluted 1:50 with 65% transmittance at 530 λ wavelength which was in accordance with the instructions given by Esteban et al.,and Fernandez Torres et al. (12,29).
The results of micro dilution tests for most strains read after 7 days at 28 °C, when adequate growth was observed in the control well with significant opacity. The 7 day time period has also been mentioned in Santos et al. (27), Gupta et al. (31),, and Barros (30) studies. This time period was shorter (4 days at 35°C) in Ghannoum et al. (10), and Mukherjee (32)) studies. This difference might be explained by the different temperatures used. Galuppi et al.(28) reported a longer period of 14 days and incubation at 30°C. This difference in the required incubation time may be due to the different volumes of fungi inoculated into the micro plates.
Antibiogram results for 320 dermatophytes showed that the highest susceptibility of dermatophytes was to itraconazole and the lowest to fluconazole. Our findings about susceptibility of dermatophytes to fluconazole is compatible withSantos et al. (27), Favre et al. (11), Barros et al. (30) and Sarifakioglu et al.(8) studies. Korting et al.(33) suggestedthat high values of MIC for fluconazole may be due to technical problems,such as interference with someingredients of the culture media or insolubility at high concentrations.
However, geometric means MIC (GM) obtained in this study showed that the results of terbinafine for all species of dermatophytes were significantly greater than results of Gupta et al,study (31), but were in agreement with Munoz et al., 16,29) findings. The GMs of dermatophytes in Favre et al., and Galuppi et al. study were greater than our study (11,28). In this study, the GMs of M. gypseum, M. canis and T. violaceum were similar (GM=0.04) and they were considered the most sensitive dermatophytes, whereas T. rubrum (GM=0.172) and T. mentagrophytes (GM=0.142) had the lowest susceptibility. In Esteban et al. (29) study,T. rubrum (GM=0.190) had the lowest susceptibility, while in Fernandez-Torres et al. study, T. rubrum and T. violaceum (GM=0.01) were the most sensitive dermatophytes (12). Considering the fact that laboratory methods were similar in these studies and also for determining the MIC endpoint for terbinafine is usually selected a well with 100% growth inhibition, it appears that the reported differences might be due to the differences between strains or different number of fungi tested.
In the present study, T. tonsurans (GM=24.24) showed the lowest and M. canis (GM=0.16) showed the highest susceptibility to griseofulv in. This is in complete agreement with Seebacher et al, opinion, and they suggest that griseofulvinis the best choice in the treatment Tinea capitis cases due to M. canis (18). T. mentagrophytes and E. floccosum have much lower susceptibility to griseofulvin than T. rubrum. Chadeganipour et al. (24) also reported higher MIC value for T. mentagrophyes than other tested dermatophytes. The GM reported by Galuppi et al. was also higher than our finding (GM=2.20) (28). However, Favre et al. reported an even smaller value (GM=0.37)(11). These differences are justifiable considering different readings/interpretations as 50%, 100% and 75% growth inhibition, respectively, compared to the growth control well of micro plate for determining MIC.
In the present study, T. mentagrophytes after M. gypseum showed the lowest and E. floccosum had the highest in-vitro sensitivity to fluconazole.
The GM in our study was much greater than the GMs obtained in Favre et al. (11),(GM=6.3) and Carrillo et al. (16), (M=15.08) study. In Carrillo et al. (16) study, M. gypseum and T. mentagrophytes had the highest GM which means the lowest susceptibility. Kotring et al. (33) showed that the concentration of this drug, following consumption of 150 mg orally after 7 days, reaches to 7.1 µg/mL in stratum corneum. In addition,MIC > 64 μg/mL is attributed to resistance to this drug (35,36). MIC mean values for half of the dermatophyes strains in our study is about 7 times greater than in-vivo condition, used for treatment of dermatophyte infections. In Foroumadi et al. (37) study, high MIC value were seen in cases of vulvovaginal candidiasis in Tehran treated with fluconazole, too.
In this study, we examined voriconazole antifugal susceptibility against dermatophytes. However, this drug is not currently being used due to its high cost. According to our study, T. schoenlinii, T. violaceum, T.verrucosum, M. canis, E. floccosum, T. rubrum, T. mentagrophytes, M. gypseum and T. tonsurans had, in an ascending order, the lowest to highest susceptibility. The GMs for all strains were greater than those obtained in Carrillo et al, Favre et al, and Fernandez -Torres et al, studies. However, Fernandez-Torres et al, and Carrillo et al, showed the highest susceptibility of E. floccosum to voriconazole (11,12,14,16,38).
Among topical antifungal agents that are usually used along with systemic antifungal medications, we selected ciclopirox olamine, clotrimazole, amorolfine and naftifine for invitro antifungal susceptibility. Amorolfine and naftifine are not available in Iran and therefore are not prescribed. In our study, M. gypseum (GM=0.176) and T. rubrum (GM=0.34) had the highest and lowest susceptibility to clotrimazole, respectively. Fernandez-Torres et al, (14)  In terms of quantity, MIC50 values in our study, except for T. tonsurans, were in agreement with Korting et al. (33) findings. The figure obtained in our study was in the range of 0.125-2 µg/mL, while this was 2 or 3 µg/mL in Korting et al, study and both these rates were greater than figures obtained by Gupta et al,Santos et al,and Favre et al. studies (11,27,31).
Susceptibility testing of dermatophytes to amorolfine has been previously performed by Li et al and Favre et al. (11,26 remains uncertain,and breakpoints with proven relevance have yet to be identified and approved by regulatory agency. Based on our results, griseofulvin and fluconazole for dermatophyte infection should be used with a greater caution. Controversial findings in our study and others point the necessity of establishing a standard method for antibiogram of dermatophytes to facilitate the selection of drug similar to what is routinely performed for yeasts and bacteria.